Much effort has been devoted to the analysis of antibodies to AIDS virus antigens, but no previous studies have defined antigenic sites of this virus which elicit T cell immunity, even though such immunity is important in protection against many other viruses. Analysis of immunodominant helper T cell cell sites has suggested that such sites tend to form amphipathic helices. Using an algorithm based on this model, two candidate T cell sites, env T1 and env T2, were identified in the human T cell lymphotropic virus type IIIb (HTLVIIIb) envelope protein that were conserved in other Human Immunodeficiency Virus (HIV) isolates. Corresponding peptides were synthesized and studied in genetically defined inbred and F.sub.1 mice for induction of lymph node proliferation. After immunization with a 425 residue recombinant envelope protein fragment, significant responses to native gp120 as well as to each peptide were observed in both F.sub.1 combinations studied. Conversely, immunization with env T1 peptide induced T cell immunity to the native gp120 envelope protein. The genetics of the response to env T1 peptide were further examined and revealed a significant response in three of four independent Major Histocompatibility haplotypes tested, an indication of high frequency responsiveness in the population.
Identification of helper T cell sites should facilitate development of a highly immunogenic, carrier-free vaccine that induces both T cell and B cell immunity. The ability to elicit T cell immunity to the native AIDS viral protein by immunization with a 16-residue peptide suggests that such sites represent potentially important components of an effective AIDS vaccine.
Since the discovery of the human immunodeficiency viruses, the causative agents of the acquired immunodeficiency syndrome (AIDS), substantial progress has been made toward characterizing the viral genes and their products in infected cells. Though responsible for profound immunodeficiency, viral infection in man consistently induces a detectable immune response as evidenced by serum antibodies to the major viral proteins. Studies of serum reactivity to specific viral proteins have revealed no consistent prognostic associations to date. Much of the host antibody response is focused on the envelope proteins gp120 and gp41 (see FIG. 1). The ability of native gp120 or a large recombinant fragment to induce neutralizing antibodies has been demonstrated. Two apparently immundominant antibody binding sites in the gp41 envelope protein have been defined at the level of small synthetic peptides. Though such antibody sites are clearly of diagnostic importance and potentially of importance for vaccine design, the typical progression of AIDS in patients despite the presence of these antibodies suggests that effective T cell immunity is important to the immune defense against this pathogen.
An ideal vaccine is highly immunogenic, induces both T cell and B cell virus-specific immunity, and is free of irrelevant carrier proteins. While traditional approaches using whole virion or virion subunits can generally achieve this, practical considerations such as safety and availability of native antigen have led many to consider more highly engineered vaccine constructs for AIDS. Localization of immunodominant T cell and B cell recognition sites becomes critical if one wishes to design a vaccine based on recombinant proteins or synthetic peptides. A T cell response to the gp120 envelope protein has been demonstrated recently by Zarling et al. Nature, 323: 344-345 (1986) in macaques immunized with vaccinia constructs containing gp120 coding sequence. However, identification and characterization of immunodominant T cell sites within this 518 residue protein or other HIV proteins have not been reported.
Antibodies typically recognize free antigen in native conformation and can potentially see almost any site exposed on the antigen surface. In contrast, typical CD4+ helper T cells recognize antigen only in the context of the class II major histocompatibility (MHC) molecule and only after appropriate antigen processing usually consisting of proteolysis or denaturation. Additionally, the polyclonal T cell response is focused only on relatively few discrete sites. This limited response is seen even for non-eukaryotic proteins (e.g., influenza hemagglutinin and staphylococcal nuclease) for which tolerance to homologous host proteins does not limit the number of antigenic sites. Therefore, it is important to find sites which do elicit T-cell immunity to AIDS viral proteins. The elucidation of features determining immunodominance residing both intrinsic and extrinsic to antigen is the focus of much current basic and clinical interest. Detailed characterization of immunodominant T cell sites has allowed exploration for general features. Such analysis led to the observation that immunodominant T cell sites tend to have an amino acid sequence consistent with formation of an amphipathic helix with hydrophilic residues on one face and hydrophobic residues on the opposite face. In an amphipathic alpha helix, the hydrophobicity varies sinusoidally with a period of 3.6 residues per turn of the helix or a frequency of 100.degree. per residue. An amphipathic 3.sub.10 helix has a period of 3 and a frequency of 120.degree.. Based on this model, an algorithm entitled AMPHI, has been developed for identification of such sequences in proteins given only primary sequence data.
Although Zarling et al., Nature 323: 344 (1986), showed that T cell immunity to the AIDS virus envelope could be induced in monkeys using a recombinant vaccinia virus carrying the gene for the whole envelope protein, their study did not identify antigenic sites stimulating these T cells. Also, vaccinia immunization has been discontinued in the U.S. because of danger of disseminated vaccinia infection and other side effects. A synthetic peptide vaccine would not carry any of these risks. Since it would be synthetic, there would be no risk of live AIDS virus contamination as might occur with a killed virus vaccine. A synthetic peptide which does not contain sites responsible for syncytia formation might produce fewer side effects than a large recombinant envelope protein containing such sites.